Hypothermic preservation of hepatocytes. II. Importance of Ca2 and amino acids

The importance of the components of a tissue culture media, Leibovitz-15 (L-15), for maintaining viability of hypothermically preserved hepatocytes was analyzed. Hepatocytes isolated from rat livers were incubated at 5 degrees C in an oxygenated environment with continuous shaking (to simulate organ perfusion preservation). L-15 + 5 g% polyethylene glycol (PEG) or variants of this solution were used as the preservation media. After 48 hr of storage, hepatocyte viability was assessed by measuring the release of LDH into the incubation medium and cell volumes were determined. Following 90 min of normothermic incubation (to simulate organ reperfusion), mitochondrial function was measured. Hepatocytes stored in the complete L-15 solution were about 90% viable at the end of 48 hr of storage, while cells stored in a solution containing only the principle electrolytes (PE) lost viability (70% viable). Only the addition of a combination of divalent cations (Ca/Mg) and amino acids was sufficient to maintain viability equivalent to that obtained in the complete L-15 mixture. Hepatocytes suspended in L-15 maintained normal cell volumes (3.85 microliters/mg protein), while cells in the PE solution were swollen with cell volumes of 4.66 microliters/mg protein. Only the addition of Ca/Mg to the PE solution was effective at suppressing cell swelling similar to the complete L-15 media. Both basal and uncoupler-stimulated respiration were depressed in cells stored in the PE solution (15 and 28 nmol O2/min/mg protein) as compared to cells in L-15 (21 and 41 nmol O2/min/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracellular calcium protects cultured rat hepatocytes from injury caused by hypothermic preservation

Effects of various preservation solutions were compared in an experimental hypothermic preservation model using cultured rat hepatocytes. Hepatocytes prepared by the collagenase perfusion method were cultured for 48 hr, then the medium in each culture dish was exchanged for various preservation solutions, and the dishes were hypothermically (0-2 degrees C) stored in a refrigerator for 12-72 hr. After the preservation period, the hepatocytes were cultured again at 37 degrees C for 2 hr. Hepatocytes' viability after 18-hr preservation and reculture was greater when they were preserved in "intracellular" rather than "extracellular" solutions. Even with Euro-Collins solution (intracellular solution), hepatocyte viability decreased to approximately 20% after 24-hr preservation, and an increase in the cellular lipid peroxide content was observed. However, when this solution contained a submillimolar concentration of calcium, lipid peroxidation was significantly suppressed and hepatocyte viability was dramatically improved. Vitamin E was almost equally effective and a marked synergistic effect was observed with calcium. Calcium was found to be capable of maintaining the cellular glutathione level during cold storage, which seems to suppress lipid peroxidation and consequently improve hepatocyte survival.     

Ischemic preconditioning in a rodent hepatocyte model of liver hypothermic preservation injury

Ischemic preconditioning (IPC) is a phenomenon of protection in various tissues from normothermic ischemic injury by previous exposure to short cycles of ischemia-reperfusion. The ability of IPC to protect hepatocytes from a model of hypothermic transplant preservation injury was tested in this study. Rat hepatocytes were subjected to 30min of warm ischemia (37 degrees C) followed by 24 or 48h of hypothermic (4 degrees C) storage in UW solution and subsequent re-oxygenation at normothermia for 1h. Studies were performed with untreated control cells and cells treated with IPC (10min anoxia followed by 10min re-oxygenation, 1 cycle). Hepatocytes exposed to IPC prior to warm ischemia released significantly less LDH and had higher ATP concentrations, relative to untreated ischemic hepatocytes. IPC significantly reduced LDH release after 24h of cold storage before reperfusion and after 48h of cold storage and after 60min of warm re-oxygenation, relative to the corresponding untreated hepatocytes. ATP levels were also significantly higher when IPC was used prior to the warm and cold ischemia-re-oxygenation protocols. In parallel studies, IPC increased new protein synthesis and lactate after cold storage and reperfusion compared to untreated cells but no differences in the patterns of protein banding were detected on electrophoresis between the groups. In conclusion, IPC significantly improves hepatocyte viability and energy metabolism in a model of hypothermic preservation injury preceded by normothermic ischemia. These protective effects on viability may be related to enhanced protein and ATP synthesis at reperfusion.     

Hypothermic preservation of hepatocytes : I. Role of cell swelling

Hepatocytes from isolated rat livers were hypothermically incubated (5 degrees C) in an oxygenated environment with continuous shaking (to simulate organ perfusion preservation). The incubation solution was either a tissue culture medium (L-15), an organ preservation perfusate (UW gluconate), or a simple cold-storage solution used for organ preservation (UW lactobionate). Hepatocyte viability was assessed from the release of lactate dehydrogenase (LDH) into the incubation medium. Cell swelling (due to the uptake of water) was also measured. Within 24 hr, hepatocytes hypothermically stored in each of the three incubation solutions became swollen (30 to 40% water gain) and lost a significant amount of LDH (as much as 60%). The addition of polyethylene glycol (PEG; relative molecular mass 8000; 5 g%) to the solutions suppressed cell swelling and allowed the incubated hepatocytes to remain relatively well preserved (30% LDH release) for as long as 120 hr. Adding either dextran (relative molecular mass 10,000 to 78,000; 5 g%) or saccharides (100 mmol/liter) instead of PEG neither prevented cell swelling nor prevented the cells from dying. The results of this study suggest (i) there is a direct correlation (r = 0.873) between hypothermia-induced cell swelling and cell death (i.e., the suppression of cell swelling prevents cell death); (ii) the mechanism by which PEG prevents cell swelling (and thus maintains cell viability) is not related to the osmotic or oncotic properties of the molecule but instead is apparently related to some unknown interaction between PEG and the cell, an interaction that provides stability during hypothermic incubation; and (iii) hypothermia-induced cell swelling must be prevented if isolated hepatocytes are to be used as a model for studying the mechanism by which cell damage occurs during hypothermic organ preservation. By eliminating cell death due to cell swelling, the biochemical mechanisms of cell death can be studied.     

Intracellular lactate dehydrogenase concentration as an index of cytotoxicity in rat hepatocyte primary culture

In searching for a reliable index for cytotoxicity testing in rat hepatocyte primary culture, lactate dehydrogenase (LDH) concentrations in lysates of attached hepatocytes and LDH released into the culture medium were compared under conditions of exposure to various dosages of sodium chloride, sodium salicylate, R-warfarin, acetaminophen, phenylbutazone, and furosemide (frusemide). The amount of intracellular LDH was assessed by inducing the cells to release the enzyme with 0.1% Tritron X-100. The induced LDH leakage was completed in 1 hr and the LDH activity was stable in storage at 10° for 2 weeks. We found that intracellular LDH is a direct indicator of the number of viable hepatocytes in contrast to the LDH released, because released LDH does not account for the significant number of cells detached from monolayer but which are not leaky, during the 6-hr test period. Based on IC₅₀ values (50% inhibitory concentration), the relative cytotoxicities are R-warfarin > phenylbutazone > furosemide > acetaminophen > sodium salicylate > sodium chloride.Abbreviations DMSO dimethyl sulfoxide - HPC hepatocyte primary culture - IC₅₀ 50% inhibitory concentration - LDH lactate dehydrogenase     

Release of Free Fatty Acids and Loss of Hill Activity by Aging Spinach Chloroplasts

The free fatty acid content of spinach chloroplasts, isolated at pH 5.8 to 8.0, has been found to vary between 3.1 and 5.5% of the total chloroplast fatty acids. When chloroplasts were incubated at room temperature for 2 hours, the free fatty acids increased by 42% and the Hill activity decreased by 70%. After 2 hours of incubation at 37 degrees , the free fatty acids increased about 3-fold and the Hill activity decreased to almost 0. The addition of crystalline bovine serum albumin largely prevented the loss of Hill activity at room temperature and at 5 degrees , but had little effect during incubation at 37 degrees . Both the release of free fatty acids and the loss of Hill activity were pH dependent. The losses were the least during incubation at pH 5.8 and the greatest during incubation at pH 8.0. The major free fatty acids released at pH 5.8 were saturated, while those released at pH 7.0 or 8.0 were mainly the unsaturated acids, alpha-linolenic acid and hexadecatrienoic acid.     

Apoptosis vs. necrosis: glutathione-mediated cell death during rewarming of rat hepatocytes

Hypothermia induces injury in its own right, but the mechanisms involved in the cell damage are still unclear. The aim of this study was to test the effects that glutathione (GSH) depletion induces on cell death in isolated rat hepatocytes, kept at 4 degrees C for 20 h, by modulating intracellular GSH concentration with diethylmaleate and buthionine sulfoximine (DEM and BSO). Untreated hepatocytes showed Annexin V stained cells (AnxV(+)), scarce propidium iodide stained cells (PI(+)) and presented a low level of lactate dehydrogenase (LDH) leakage after 20 h at 4 degrees C and rewarming at 37 degrees C. When DEM and BSO were added before cold storage, we observed a few AnXV(+) cells and an increase in PI(+) cells associated with LDH release in the incubation medium. Conversely, the addition of DEM and BSO only during rewarming caused a marked increase in cell death by apoptosis. Production of reactive oxygen species (ROS) and thiobarbituric acid species (TBARS), associated with a decrease in GSH concentrations, was higher when DEM and BSO were added before cold storage. Cells treated with DEM and BSO before cold storage showed lower ATP energy stores than hepatocytes treated with DEM and BSO only during rewarming. Pretreatment of hepatocytes with deferoxamine protected against apoptotic and necrotic morphology in conditions of GSH depletion. These results suggest that pretreatment of hepatocytes with DEM and BSO before cold storage induces necrosis, while the treatment of hepatocytes only during rewarming increases apoptosis. In both conditions, iron represents a crucial mediator of cell death.     

Iron-dependent vs. iron-independent cold-induced injury to cultured rat hepatocytes: a comparative study in physiological media and organ preservation solutions

We previously described the entity of cold-induced apoptosis to rat hepatocytes and characterized its major, iron-dependent pathway. However, after cold incubation in some solutions, e.g. cell culture medium, hepatocytes show an additional, yet uncharacterized component of cold-induced injury. We here assessed the effects of organ preservation solutions on both components of cold-induced injury and tried to further characterize the iron-independent component. None of the preservation solutions (University of Wisconsin, histidine-tryptophan-ketoglutarate, Euro-Collins, histidine-lactobionate, sodium-lactobionate-sucrose and Celsior solutions) provided significant protection against cold-induced cell injury (LDH release after 24-h cold incubation/3h rewarming >65% for all solutions); three solutions even enhanced cold-induced injury. However, when the predominant iron-dependent mechanism was eliminated by the addition of iron chelators, all preservation solutions yielded hepatocyte protection that was clearly superior to the one obtainable in cell culture medium or Krebs-Henseleit buffer with iron chelators (LDH release after 24-h cold incubation/3h rewarming 相似文献   

Effects of storage and incubation conditions on human granulocyte phagocytic, bactericidal, and chemotactic functions

This study was undertaken to determine the effects of different incubation conditions on human granulocyte (PMN) bactericidal, phagocytic, and chemotactic functions. Specifically, (1) how long may a patient's blood be held before assay and maintain original PMN function, and (2) how long may isolated PMNs be incubated for the purpose of exposure to various agents and still maintain original function? PMNs isolated following storage of whole heparinized blood at 4 degrees C for 24 and 48 hr phagocytized as well as fresh cells and their bactericidal activity was 96 and 85% of control values after 24 and 48 hr, respectively. Chemotaxis decreased to 62% of control after 24 hr. The bactericidal capacity of isolated PMNs stored at 4 degrees C for 24, 48, and 72 hr decreased to 85, 81, and 78% of controls, respectively. Phagocytosis after 24 hr storage was equal to controls. Chemotaxis was decreased to 59 and 34% of controls after 24 and 48 hr, respectively. Isolated PMNs incubated at 37 degrees C demonstrated impairment in phagocytic capacity after only 4 hr.     

Glycine prevention of cold ischemic injury in isolated hepatocytes

Isolated hepatocytes suspended in a liver preservation solution (University of Wisconsin (UW) solution) and exposed to cold (5 degrees C) ischemia lose viability (LDH release) after 3 (76.5 +/- 2.6% extracellular LDH) and 4 days (90.3 +/- 5.7% extracellular LDH) storage when rewarmed (37 degrees C) in Krebs-Henseleit buffer. However, if 3 mM glycine is added to Krebs-Henseleit buffer the loss of LDH on rewarming was suppressed (% LDH = 24.4 +/- 2.2% and 33.2 +/- 3.0%, at 3 and 4 days, respectively). The protection by glycine could also be obtained by storing the hepatocytes in the UW solution containing 15 mM glycine and rewarming in the absence of glycine in Krebs-Henseleit buffer. There did not appear to be a relationship between the protection by glycine and glutathione concentration of the hepatocytes as shown by the lack of effect of a glutathione synthetase inhibitor (butathionine sulfoximine) on the protective effects of glycine. Other amino acids did not provide protection to hepatocytes exposed to cold ischemia. The mechanism of action of glycine is not known, but this compound may be important in improving cold storage of livers for transplantation.     

The effect of carbon tetrachloride on isolated rat hepatocytes

Isolated rat hepatocytes were incubated with carbon tetrachloride (CCl4) at a concentration of 0.2 mol CCl4/ml of incubation medium. The ultrastructural alterations and release of lactate dehydrogenase (LDH) and glutamate-oxaloacetate transaminase (GOT), were recorded after different periods of incubation. After 5 min incubation with CCl4, morphological changes observed by electron microscopy, involved the plasma membrane. The endoplasmic reticulum and mitochondria were altered later. These morphological alterations were accompanied by an early release of LDH and GOT into the incubation medium. It is concluded that, in contrast with its in vivo effects, in vitro CCl4 can induced an early morphological alteration of the hepatocyte plasma membrane before damaging the endoplasmic reticulum.     

Effect of polyethylene glycol on lipid peroxidation in cold-stored rat hepatocytes

A mechanism suggested to cause injury to preserved organs is the generation of oxygen free radicals either during the cold-storage period or after transplantation (reperfusion). Oxygen free radicals can cause peroxidation of lipids and alter the structural and functional properties of the cell membranes. Methods to suppress generation of oxygen free radicals of suppression of lipid peroxidation may lead to improved methods of organ preservation. In this study we determined how cold storage of rat hepatocytes affected lipid peroxidation by measuring thiobarbituric acid reactive products (malondialdehyde, MDA). Hepatocytes were stored in the UW solution +/- glutathione (GSH) or +/- polyethylene glycol (PEG) for up to 96 h and rewarmed (resuspended in a physiologically balanced saline solution and incubated at 37 degrees C under an atmosphere of oxygen) after each day of storage. Hepatocytes rewarmed after storage in the UW solution not containing PEG or GSH showed a nearly linear increase in MDA production with time of storage and contained 1.618 +/- 0.731 nmol MDA/mg protein after 96 h. When the storage solution contained PEG and GSH there was no significant increase in MDA production after up to 72 h of storage and at 96 h MDA was 0.827 +/- 0.564 nmol/mg protein. When freshly isolated hepatocytes were incubated (37 degrees C) in the presence of iron (160 microM) MDA formation was maximally stimulated (3.314 +/- 0.941 nmol/mg protein). When hepatocytes were stored in the presence of PEG there was a decrease in the capability of iron to maximally stimulate lipid peroxidation. The decrease in iron-stimulated MDA production was dependent upon the time of storage in PEG (1.773 nmol/mg protein at 24 h and 0.752 nmol/mg protein at 48 h).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of phospholipase A2 inhibitors on the release of arachidonic acid and cell viability in cold-stored hepatocytes

We investigated the effect of phospholipase A(2) (PLA(2)) inhibitors on PLA(2) activity and cell viability in cold-stored rat hepatocytes. The cells were radiolabeled with [(3)H] arachidonic acid (AA) and cold stored in the University of Wisconsin (UW) solution containing various PLA(2) inhibitors. PLA(2) activity was determined by measuring the total free (cellular + supernatant) AA by thin-layer chromatography after inhibiting reacylation of free AA with inhibitors of energy production (carbonyl cyanide m-chlorophenylhydrazone + iodoacetate). Aristolochic acid, chlorpromazine, and quinacrine in the UW solution showed a significant inhibitory effect throughout 48 h cold storage but only at relatively high concentration. PLA(2) activity was also suppressed (58% of control) by trifluoperazine (50 microM), but its effect was limited to only 24 h. In contrast, pretreatment of the cells prior to hypothermic preservation with trifluoperazine (10 to 100 microM) suppressed PLA(2) activity during 48 h storage. Inclusion of calmodulin antagonist W-7 did not affect PLA(2) activity. Thus, the inhibitory activity of these agents appears unrelated to Ca-calmodulin-phospholipid interaction but to have an inhibitory effect on PLA(2) activity. To study the effects of PLA(2) inhibitors on cell viability, lactate dehydrogenase (LDH) release was measured in the presence or absence of inhibitors upon rewarming cold-stored cells in Krebs-Henseleit buffer for 2 h at 37 degrees C. None of the inhibitors tested improved cell viability after 48 h storage. Thus, although PLA(2) inhibitors blocked PLA(2) activity, there was no suppression of LDH release. PLA(2) may play a minor role in preservation/reperfusion injury to cold-stored hepatocytes.     

Motility and fertility of alginate encapsulated boar spermatozoa

Ejaculated boar spermatozoa are vulnerable to cold shock. Prolonged storage of boar spermatozoa at low temperatures reduces survival rate, resulting in a bottleneck for the extension of artificial insemination in pig husbandry. This study evaluated whether alginate microencapsulization processing can improve the longevity of boar spermatozoa stored at 5 degrees C and the fertility of microencapsulated spermatozoa in vivo. Sperm-rich fraction semen from three purebred boars were concentrated and microencapsulated using alginate at 16-18 degrees C, and then were stored at 5 degrees C. Following storage for 1, 3 and 7 days, the microcapsule was taken out to assess sperm release under 37 degrees C incubation with or without 110 rpm stirring. The percentage of sperm released from microcapsules with 110 rpm stirring was higher than without stirring (81 versus 60%) after 24h of incubation. In another experiment, semen was also microencapsulated to evaluate the sperm motility. The motility of spermatozoa was assessed at 10 min, 8, 24, 32, 48, 56 and 72 h following incubation at 37 degrees C for nine consecutive days. The fertility of the free and microencapsulated semen was assessed by inseminating sows, and the reproductive traits (conception rate, farrowing rate, and litter size) were recorded. The motility of encapsulated spermatozoa was significantly higher than that of free semen after 8h incubation at 37 degrees C after storing for over three days (P<0.05). No significant difference existed in conception rate, farrowing rate, and litter size between the microencapsulated and non-encapsulated semen after four days of storage. In conclusion, microencapsulation can increase the longevity of boar spermatozoa and may sustain in vivo ova fertilization ability.     

Cold storage of rat hepatocyte suspensions for one week in a customized cold storage solution--preservation of cell attachment and metabolism

Background & Aims

Primary hepatocytes are of great importance for basic research as well as cell transplantation. However, their stability, especially in suspension, is very low. This feature severely compromises storage and shipment. Based on previous studies with adherent cells, we here assessed cold storage injury in rat hepatocyte suspensions and aimed to find a cold storage solution that preserves viability, attachment ability and functionality of these cells.

Methods

Rat hepatocyte suspensions were stored in cell culture medium, organ preservation solutions and modified TiProtec solutions at 4°C for one week. Viability and cell volume were determined by flow cytometry. Thereafter, cells were seeded and density and metabolic capacity (reductive metabolism, forskolin-induced glucose release, urea production) of adherent cells were assessed.

Results

Cold storage injury in hepatocyte suspensions became evident as cell death occurring during cold storage or rewarming or as loss of attachment ability. Cell death during cold storage was not dependent on cell swelling and was almost completely inhibited in the presence of glycine and L-alanine. Cell attachment could be greatly improved by use of chloride-poor solutions and addition of iron chelators. Using a chloride-poor, potassium-rich storage solution containing glycine, alanine and iron chelators, cultures with 75% of the density of control cultures and with practically normal cell metabolism could be obtained after one week of cold storage.

Conclusion

In the solution presented here, cold storage injury of hepatocyte suspensions, differing from that of adherent hepatocytes, was effectively inhibited. The components which acted on the different injurious processes were identified.     

Influence of Temperature of Incubation and Type of Growth Medium on Pigmentation in Serratia marcescens

Maximal amounts of prodigiosin were synthesized in either minimal or complete medium after incubation of cultures at 27 C for 7 days. Biosynthesis of prodigiosin began earlier and the range of temperature for formation was greater in complete medium. No prodigiosin was formed in either medium when cultures were incubated at 38 C; however, after a shift to 27 C, pigmentation ensued, provided the period of incubation at 38 C was not longer than 36 hr for minimal medium or 48 hr for complete medium. Washed, nonpigmented cells grown in either medium at 38 C for 72 hr could synthesize prodigiosin when suspended in saline at 27 C when casein hydrolysate was added. These suspensions produced less prodigiosin at a slower rate than did cultures growing in casein hydrolysate at 27 C without prior incubation at 38 C. Optimal concentration of casein hydrolysate for pigment formation by suspensions was 0.4%; optimal temperature was 27 C. Anaerobic incubation, shift back to 38 C, killing cells by heating, or chloramphenicol (25 mug/ml) inhibited pigmentation. Suspensions of washed cells forming pigment reached pH 8.0 to 8.3 rapidly and maintained this pH throughout incubation for 7 days. Measurements of viable count and of protein, plus other data, indicated that cellular multiplication did not occur in suspensions of washed cells during pigment formation. By this procedure utilizing a shift down in temperature, biosynthesis of prodigiosin by washed cells could be separated from multiplication of bacteria.     

Effects of 2-hydroxypropyl-beta-cyclodextrin and cholesterol on porcine sperm viability and capacitation status following cold shock or incubation

Porcine sperm are extremely sensitive to the damaging effects of cold shock. It has been shown that cholesterol-binding molecules, such as 2-hydroxypropyl-beta-cyclodextrin (HBCD), improve post-cooling porcine sperm viability when added to an egg yolk-based extender, but also enhance sperm capacitation in other species. The objective of this study was to determine the effects of HBCD and cholesterol 3-sulfate (ChS) on porcine sperm viability and capacitation following cold shock or incubation under conditions that support capacitation using a defined medium. We report here that porcine sperm incubated in medium containing both HBCD and ChS have significantly improved viability following cold shock (10 min at 10 degrees C) when compared to sperm incubated without HBCD or ChS, or with either component alone. Treatment with HBCD plus ChS also completely inhibited the increase in protein tyrosine phosphorylation induced by the cold shock treatment or by incubation for 3 hr under conditions that support capacitation. Two assays of sperm capacitation, the rate of calcium ionophore-induced acrosome reactions and chlortetracycline (CTC) staining, were not significantly altered by HBCD and ChS following cold shock. However, 3-hr incubation with HBCD plus ChS or with 1 mM ChS alone decreased the percentage of sperm undergoing the induced acrosome reaction without significantly affecting viability when compared to the control. These results indicate that the manipulation of sperm plasma membrane cholesterol content affects porcine sperm viability and capacitation status and could therefore be useful to protect sperm from cold shock during cryopreservation by improving viability without promoting premature capacitation.     

Drug metabolism and viability studies in cryopreserved rat hepatocytes

Rat hepatocytes were cryopreserved optimally by freezing them at 1 degrees C/min to -80 degrees C in cryoprotectant medium containing either 20% (v/v) dimethylsulfoxide (Me2SO) and 25% (v/v) fetal calf serum in Leibowitz L15 medium (Me2SO cryoprotectant) or 25% (v/v) vitrification solution (containing Me2SO, acetamide, propylene glycol and polyethylene glycol) in Leibowitz L15 medium (VS25). The VS25 solution was superior for maintaining viability during short-term storage (24-48 hr) but was slightly toxic during longer storage periods (7 days). Although thawed cells were 40-50% viable on ice after cryopreservation, their viability fell rapidly during incubation in suspension at 37 degrees C. This decline in viability occurred more rapidly after freezing in Me2SO cryoprotectant than in VS25 and was associated with extensive intracellular damage and cell swelling. The loss in viability at 37 degrees C does not appear to be due to ice-crystal damage as it occurred in cells stored at -10 degrees C (above the freezing point of the cryoprotectants) and it may be due to temperature/osmotic shock. Both cryoprotectant media were equally efficient at preserving enzyme activities in the hepatocytes over 7 days at -80 degrees C. Cytochrome P450 and reduced glutathione content and the activities of the microsomal enzymes responsible for aminopyrine N-demethylation and epoxide hydrolysis were well maintained over 7 days storage. In contrast, the cytosolic enzymes glutathione-S-transferase and glutathione reductase were markedly labile during cryopreservation. Cytosolic enzymes may be more susceptible to ice-crystal damage, whereas the microsomal membrane may protect the enzymes which are embedded in it.     

Storage of Chinchilla lanigera Semen at 4°C for 24 or 72 h with Two Different Cryoprotectants

The objectives of this study were to: (a) test the functional activity of Chinchilla lanigera spermatozoa suspended in either glycerol or ethylene glycol, cooled to 4 degrees C, and stored for 24 or 72 h and (b) investigate, after these cooling periods, the effects of incubating sperm at 37 degrees C (for 4 h) upon sperm functional activity. The ejaculate was mixed with the cryoprotectant medium (at 1 M final concentration) and cooled to 4 degrees C. After warming, sperm motility, sperm viability, hypoosmotic swelling test results, and acrosomal integrity were significantly higher for samples containing ethylene glycol than for those in glycerol, stored for 24 or 72 h, and then assayed after 0 or 4 h incubation at 37 degrees C. A significant reduction of sperm motility and viability was detected only when the glycerol cryoprotectant agent was employed, compared to the fresh samples. These results clearly indicate that under our experimental conditions, ethylene glycol is a better protectant for sperm storage than glycerol.     

Inhibition of microcystin-induced release of cyclooxygenase products from rat hepatocytes by anti-inflammatory steroids

We showed previously that exposure to microcystin causes eicosanoid release. That study was extended further to test the effect of glucocorticoids on microcystin-induced release of [14C]arachidonic acid and its metabolites from rat hepatocytes previously treated with [14C]arachidonic acid. Release of total radioactivity was 4-fold greater from hepatocytes after 2-hr incubation with 1 microM microcystin than after incubation with control medium. Fluocinolone pretreatment decreased the microcystin-induced synthesis and release of prostacyclin by 24 +/- 2.6% (P less than 0.05) and thromboxane B2 by 39 +/- 3% (P less than 0.025). Treatment of hepatocyte cultures with either microcystin (1 microM) or steroids had no effect on cell viability or total cell protein. Total radioactivity released into the incubation medium was not affected by glucocorticoid alone. Under these conditions, the quantities of both prostaglandin F2 alpha and prostaglandin E2 released were not significantly different when control and microcystin-treated cultures were compared. The half-maximal inhibition (IC50) values obtained from the dose-response data for the inhibition of arachidonic acid release by steroids were comparable with normal cortisol levels in humans. Dose-response curves gave the following rank order of inhibitory potency: fluocinolone greater than dexamethasone greater than hydrocortisone. These results suggest that glucocorticoid therapy might be beneficial in microcystin toxicosis.